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calibration standards and as retention time markers. The seven- point calibration concentration levels for the MGRS are listed in Table 2018.09B . D. Preparation of Calibration Standard Solutions Note: If not used immediately, all solutions should be stored at –20°C. When stored at 2–8°C, solutions are stable for 24 h. All solutions are diluted with HPLC grade methanol and mixed well. Note: Transfer solutions into microliter HPLC vial inserts with a pipet. Ensure that there are no air bubbles in the insert prior to injection. ( a )  Linearity 1 .—Pipet 50 uL MGRS into an HPLC vial. ( b )  Linearity 2 .—Pipet 400 µL MGRS and dilute the solution with 800 µL HPLC grade methanol. ( c )  Linearity 3 .—Dilute 700 µL Linearity 2 solution with 500 µL HPLC grade methanol. ( d )  Linearity 4 .—Dilute 700 µL Linearity 3 solution with 500 µL HPLC grade methanol. ( e )  Linearity 4a .—Dilute 700 µL Linearity 4 solution with 500 µL HPLC grade methanol. Note : This is only used to make Linearity 6, not for instrument calibration. ( f )  Linearity 5 .—Dilute 700 µL Linearity 4a solution with 500 µL HPLC grade methanol. ( g )  Linearity 6 .—Dilute 700 µL Linearity 5 solution with 500 µL HPLC grade methanol. ( h )  Linearity 7 .—Dilute 700 µL Linearity 6 solution with 500 µL HPLC grade methanol. E. Preparation of Sample Test Solutions ( a )  Powdered raw materials and powdered extracts.— ( 1 ) Accurately weigh all samples into 50 mL conical tubes. Use the following weights (±0.1 mg) and codes for each individual ginseng matrix: GR No. for P. ginseng/P. quinquefolius whole root samples (400 mg) and GE No. for P. ginseng/P. quinquefolius powdered extracts (200 mg). ( 2 ) Add 10 mL extraction solvent. Mix the sample tube using a vortex mixer for at least 1 min to ensure optimal mixing, and then sonicate for 25 min at room temperature. ( 3 ) Centrifuge the sample tubes at 3500 rpm for 5 min. ( 4 ) Decant the solvent into a prelabeled 25 mL volumetric flask and identically label the flask with its appropriate sample identification code. ( 5 ) Repeat steps ( 2 ) to ( 4 ) with a second 10 mL aliquot extraction solvent. ( 6 ) Repeat steps ( 2 ) to ( 4 ) a third time using 4 mL extraction solvent. Combine the extracts. ( 7 ) Rinse the extracted residue with approximately 1 mL extraction solvent and vortex mix for 5 s. Centrifuge at 3500 rpm for 5 min, decant into the same volumetric flask, and bring to a final volume of 25 mL using the extraction solvent. ( 8 ) Cap and invert the volumetric flask at least 10 times to ensure complete mixing of the extract solution. Note: For root samples, follow steps ( 9 ) to ( 11 ). For powdered extracts and finished products (GC and GT below) proceed directly to step ( 11 ). ( 9 ) Pipet 1 mL extract solution into a 2 mL microcentrifuge tube and add 100 µL base hydrolyzing agent (5% KOH, w/v). Mix solution using a vortex mixer. Cover and allow the mixture to sit for 2 h at room temperature protected from direct light. ( 10 ) Neutralize solution by adding 100 µL neutralizing agent (14%KH 2 PO 4 , w/v) solution. Mix the solution using a vortex mixer.

( 11 ) Transfer an aliquot of the solution into an HPLC vial using a syringe fitted with a 0.45 µm PTFE filter. ( b )  Hard-filled capsules.— ( 1 ) Determine total capsule content weight ( C ) by weighing 20 capsules. Record weight. ( 2 ) Cut open the 20 capsules with a suitable cutting instrument. ( 3 ) Empty and combine the capsules’ contents in a conical tube. Record weight of the capsules’ contents, as well as weight of the empty capsule shells ( S ). Average capsule fill weight is calculated using the following equation: Average capsule fill weight, g = ( C – S )/20 ( 4 ) Record the average capsule fill weight. ( 5 ) Transfer the contents into a 50 mL conical tube and mix using a spatula to homogenize the samples. ( 6 ) Accurately weigh the sample into a 50 mL conical tube. Use the following weight (±0.1 mg) and code for this ginseng matrix: GC No. for P. ginseng/P. quinquefolius capsule (400 mg). ( 7 ) Follow the same procedure as indicated for powdered raw materials and finished products in ( a ) from steps ( 2 ) to ( 8 ), then ( 11 ). The basic hydrolysis step is not necessary for this sample type. ( c )  Tablets and caplets.— ( 1 ) Determine the average tablet weight by weighing out 20 tablets or caplets. The average weight is calculated using the following equation: Average tablet or caplet weight, g = (total weight)/20 ( 2 ) Grind the materials to make a homogeneous fine powder using a mortar and pestle, and then transfer the contents into a 50 mL conical tube. Mix using a spatula to homogenize the samples. ( 3 ) Accurately weigh the sample into a 50 mL conical tube. Use the following weight (±0.1 mg) and code for this sample type: GT No. for P. ginseng / P. quinquefolius tablets (1000 mg). ( 4 ) Follow the same procedure as indicated for powdered raw materials and finished products in ( a ) from steps ( 2 ) to ( 8 ), then ( 11 ). Note that basic hydrolysis is not necessary for this sample type. F. Determination ( a )  System suitability test .—Equilibrate the HPLC system with the mobile phase for at least 5 min until a stable baseline is obtained. Inject 10 µL mixed standard solution eight times (e.g., Linearity 7, lowest concentration). Repeatability relative standard deviation (RSD r ) of ≤5% for peak area, reference analytes chromatographically resolved (R s ) ≥ 1.5, and peak tailing ( T ) ≤ 2 are desirable for the system to be suitable for the analysis.

Table 2018.09C. Approximate retention times of the six major ginsenosides

Ginsenoside

Retention time, min

Rg1

16.0–16.4 16.5–16.9 28.7–29.0 29.0–30.1 31.3–31.7 34.4–34.8

Re

Rb1

Rc

Rb2

Rd

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